Common loss of far-red light photoacclimation in cyanobacteria from hot and cold deserts: a case study in the Chroococcidiopsidales

Deserts represent an extreme challenge for photosynthetic life. Despite their aridity, they are often inhabited by diverse microscopic communities of cyanobacteria. These organisms are commonly found in lithic habitats, where they are partially sheltered from extremes of temperature and UV radiation. However, living under the rock surface imposes additional constraints, such as limited light availability, and enrichment of longer wavelengths than are typically usable for oxygenic photosynthesis. Some cyanobacteria from the genus Chroococcidiopsis can use this light to photosynthesize, in a process known as far-red light photoacclimation, or FaRLiP. This genus has commonly been reported from both hot and cold deserts. However, not all Chroococcidiopsis strains carry FaRLiP genes, thus motivating our study into the interplay between FaRLiP and extreme lithic environments. The abundance of sequence data and strains provided the necessary material for an in-depth phylogenetic study, involving spectroscopy, microscopy, and determination of pigment composition, as well as gene and genome analyses. Pigment analyses revealed the presence of red-shifted chlorophylls d and f in all FaRLiP strains tested. In addition, eight genus-level taxa were defined within the encompassing Chroococcidiopsidales, clarifying the phylogeny of this long-standing polyphyletic order. FaRLiP is near universally present in a generalist genus identified in a wide variety of environments, Chroococcidiopsis sensu stricto, while it is rare or absent in closely related, extremophile taxa, including those preferentially inhabiting deserts. This likely reflects the evolutionary process of gene loss in specialist lineages.

. Strains used in the study.Multiple pieces of evidence show their capacity to either acclimate to far-red light (blue) or not (red).'apcE2' refers to the apcE2 marker gene PCR assay; 'CLSM' is confocal laser scanning microscopy.Most strains which tested positive for the marker gene also showed the presence of red-shifted chlorophylls, but a minority (e.g.strains CCALA 43 and 44) did not complete the transition.It is possible that this could be affected by other organisms competing in non-axenic cultures.a BCCM/ULC (University of Liège, Belgium), CCALA (Culture Collection of Autotrophic Organisms, Czech Republic), CCMEE (Culture Collection of Microorganisms from Extreme Environments), PCC (Pasteur Culture Collection of Cyanobacteria, France), SAG (Culture Collection of Algae at the University of Göttingen, Germany).b Sampling environments were categorized into hot deserts (H), cold deserts (C), hotand-cold deserts (HC) and non-extreme (NE).c C. cf cubana CCALA 45 is equivalent to C. cubana SAG 39.79.d Data for C. thermalis PCC 7203 and C. cubana SAG 39.79 can be found in previous work [1,2].e While originally Chroococcidiopsis sp.SAG 2026 was tested positive for all markers listed, it was discovered that the lab strain used was a duplicate of Chroococcidiopsis sp.SAG 2025.Strain Bü 96.1 (origin of, and equivalent to SAG 2026) was tested in its place and proved negative.

Primers
Sequence (5' to  S4.Average similarity of 16S rRNA gene sequences between different Chroococcidiopsidales clades under study, or within the same clade.For the outgroup (Pleurocapsa), only one sequence was used (Pleurocapsa sp.PCC 7327).Clades numbered as shown in Figures 3-4. a Two additional groups were used in this analysis, which represent the sister-clades to Chroococcidiopsis sensu stricto (I) and Hot desert Chroococcidiopsidales (II) in these figures, with low support values.Within clades, any two sequences share on average >95% similarity (blue).Between clades, this average typically falls to 90-92% (yellow) or even <90% (red) for highly divergent groups.This supports the assignment of these clades as separate genera [7].S5.Chroococcidiopsidales clades in scientific literature.a "Sister-clade to (I)" and "Sister-clade to (II)" appear to be sister clades to Chroococcidiopsis sensu stricto (I) and Hot desert Chroococcidiopsidales (II), respectively, and may be part of the same genera.They were listed separately due to low bootstrap support at the branching point.b The hot-wet Chroococcidiopsis clade in Pointing et al., 2007 might be equivalent to the 'Additional desert clade' (97% similarity between some sequences), but the short sequences make it difficult to confirm.

Hot desert
Chroococcidiopsidales (II) a  S6.Summary of FaRLiP distribution in the Chroococcidiopsidales.The numbers include strains tested in this study, in a previous study [13], and those with a complete genome available where the presence/absence of the FaRLiP cluster can be confirmed.Numbers in parentheses also consider fragmented genomes.a Two of the positive Hot desert Chroococcidiopsidales strains were identical in 16S rRNA.b   S7.NCBI accession numbers.The entries listed in red have been submitted to the NCBI database in the course of this study.If the sequences recovered proved identical to/shorter than existing data, they were not submitted.Some strains (CCMEE 88, 130, 313) carry two versions of the ITS, with the longer variant including tRNA-Ala and tRNA-Ile genes.

Figure S1 .
Figure S1.Capacity for FaRLiP in the Chroococcidiopsidales, as assessed by confocal microscopy.All strains contain chlorophyll a and phycobilisomes, as shown in magenta (fluorescence emission range 660-700 nm).Strains undergoing FaRLiP also show fluorescence around 720-780 nm (yellow), indicating the presence of chlorophylls d/f.FaRLiP-positive strains are marked with yellow bands; negative strains are marked with magenta bands.A minority of mixed or transitional results were also observed (mixed bands).The rightmost column shows a λ (wavelength)-scan for a minimum of 3 cells.Fluorescence emission channels are highlighted in grey bars.

Figure S2 .
Figure S2.A 16S rRNA gene phylogeny of the Hot desert Chroococcidiopsidales (II), previously 'Hot desert Chroococcidiopsis.They are preferentially found as endoliths in arid, hot environments.Tree rooted with Chroococcidiopsis thermalis PCC 7203 as outgroup.Only three strains tested positive for FaRLiP (red highlight), while the majority of tested strains were negative (black squares).This is in contrast to the Chroococcidiopsis sensu stricto genus.Strains CCMEE 10 and CCMEE 12 were grouped together due to identical 16S rRNA gene sequences (I).Tree built with RaxML.Bootstrap values < 30 not shown.

Figure S3 .
Figure S3.Network visualization of the main species clusters within the Chroococcidiopsidales.While not implying evolutionary relationships on its own, this sequence similarity-based network of the 16S rRNA gene alignment is fairly consistent with the corresponding phylogenetic tree (Figure 3).Same taxa were used.Filled open circles mark type strains, where available.The open red circle marks the distantly-related Pleurocapsa sp.PCC 7327.Minor clades Haliplanktos (VII) and Pseudocyanosarcina (VIII) are not shown.Default settings (NeighborNet, SplitsNetworkAlgorithm).

Figure S4 .
Figure S4.A 16S rRNA gene phylogeny of strains related to Gloeocapsa (III).They are preferentially found in hot springs or saline environments.The following genera have been defined: Gloeocapsa (A), Chroogloeocystis (B), Gloeocapsopsis (C), Spelotes (D).Tree rooted with Chroococcidiopsis thermalis PCC 7203 as outgroup.Black squares marks strains which tested negative for FaRLiP, either in the laboratory (CCALA 703) or by lacking the necessary genes in complete genomes.Tree built with RaxML.Bootstrap values < 30 not shown.

Figure S5 .
Figure S5.A 16S rRNA gene phylogeny of Aliterella (VI).These strains are often found in cold deserts, but are not limited to them.The tree was rooted with Chroococcidiopsis thermalis PCC 7203 as outgroup.Unlike the outgroup, none of the strains tested showed the capacity for FaRLiP (black squares).Built with RaxML.Bootstrap values <30 not shown.

Figure S6 .
Figure S6.A 16S rRNA gene phylogeny of a yet-unnamed Chroococcidiopsidales taxon referred in this work as 'Additional desert clade' (V).No known strains from culture collections are present in this group, but 16S rRNA gene similarity hints at it being a distinct genus.The tree was rooted with Chroococcidiopsis thermalis PCC 7203 as outgroup.Built with RaxML.Bootstrap values <30 not shown.

Figure S11 .
Figure S11.Venn diagram illustrating shared groups of orthologous genes (orthogroups) within the Chroococcidiopsidales.There are more clade-specific orthogroups (in bold) than orthogroups shared between any two clades.Only one incomplete MAG (ignimbrite12) was available for clade II (Hot desert Chroococcidiopsidales).This likely explains the fewer clade-specific genes recovered.Created with OrthoVenn2.

Figure S12 .
Figure S12.Legend and brief guidelines for the expanded online version of Figure 4, which can be accessed at https://itol.embl.de/tree/1301336668200071662129117.

Table S2 .
Primers used in the study.

Table S3 .
Statistics for assembled genomes and metagenome bins.Where possible, these were submitted to NCBI.Low sequencing depth for SAG 2023 resulted in a large number of indels, and as such this genome will be made available on figshare instead, together with the low-completion ignimbrite MAGs.Figshare repository: https://figshare.com/projects/Chroococcidiopsis-related_metagenomic_data/149038.

Table S8 .
Revised taxonomic assignment of strains labeled as Chroococcidiopsis whose 16S rRNA sequences are very different from Chroococcidiopsis thermalis PCC 7203 (<90% similarity).They were recovered by the use of standard filters in web BLAST.Maximum identity represents the highest identity score when the sequence is queried against the non-redundant NCBI database.Sequences labeled as 'uncultured' were not considered.smalland large nitrite reductase component (dissimilatory); gene from assimilatory pathway shared with 'Gloeocapsa', nitric oxide synthase oxygenase (shared with C. cubana)

Table S9 .
A subset of clade-specific genes highlighted by OrthoVenn2.Putative pathways were identified with BlastKOALA and KofamKOALA on the KEGG webserver.Putative functions were identified with the above in conjunction with standard BLAST.For the Hot desert Chroococcidiopsidales clade (II), there was no published genome available, only a metagenomic bin.As such, the genes in question are listed in TableS10.Asterisks mark genes which have few to no homologues in other cyanobacteria (as judged from BLAST output).

Table S10 .
Sequences specific to clade II (Hot desert Chroococcidiopsidales), recovered from the previously-published ignimbrite12 metagenome.Putative functions were identified with BlastKOALA/ KofamKOALA and BLAST.